Plan and Goals: The long-term objective of the proposed research is to discover how iron in Rieske non-heme iron oxygenases and in cytochromes P450 oxygenases activates oxygen for reactions with substrates. The specific aims focus on characterizing intermediates in these reactions by rapid kinetics and other physical means. We will employ stopped-flow kinetics and rapid freeze-quench methods, coupled with spectroscopy, including UV-visible, EPR, ENDOR, and Mossbauer methods, to characterize intermediates occurring in the reaction. Double-mixing methods, whereby a transient intermediate is formed and then reacted with a substrate or other chemical agent at a defined time, will be used to examine the reactivity of intermediates observed. Our recent studies have elucidated conditions that will enable us to maximize the formation of three high-valent intermediates with cytochrome P450cam (compound I, compound ll-like species, and compound ES). We now plan to investigate these species by the methods mentioned above. We believe that similar approaches will likewise be useful for studying phthalate dioxygenase, one of the two best-characterized Rieske oxygenases that catalyze the first step in the microbial aerobic metabolism of many aromatic compounds. Relevance to Public Health: Oxygenases are found in all aerobic organisms and are important in the biosynthesis, transformation, and degradation of steroids, nucleic acids, catecholamines, collagen, drugs, prostaglandins, lignin, and various foreign compounds. Thus, these enzymes are crucial to a majority of aerobic life forms and are requisite to the development of bioremediation processes necessary for dealing with pollution in our environment, perhaps the most serious long-term health problem of the world. In addition to their role in biodegradation, the products of Rieske nonheme iron-containing enzymes are often c/s-dihydrodiols, which are valuable in "green" synthetic chemistry that seeks to minimize chemical pollution and its effects on health. P450 enzymes are critical in the metabolism of drugs and other xenobiotic substances. Thus, an understanding of their function is necessary for developing effective pharmaceutical products. We believe that results from these studies will lead to a better comprehension of the electronic structure of intermediates involved in oxygenation reactions, and this will be important in understanding how molecular oxygen is activated for controlled metabolic processes. This may, in turn, lead to an improved ability to predict and deal with the metabolism of various compounds in the environment.